The mushroom species Agaricus bisporus (Lange) Imbach, also known as Agaricus brunnescens Peck, is a well known and widely cultivated commercial mushroom. At least one distinct horticultural variety, i.e., cultivated strain, of this species of mushroom has been the subject of a U.S. Plant Patent, No. Plant 7,636, incorporated herein by reference.
Notably, a distinctive characteristic of Agaricus bisporus, that historically has defined the species, is that virtually all previously known strains have predominantly produced only two spores on each basidium. Generally, only small percentages (less than 10 percent) of basidia having more than two spores have been shown to occur in various Agaricus bisporus strains. In very rare instances, laboratory strains derived from predominantly bisporic strains have been reported to exhibit basidia in which basidia bearing more than two spores may predominate. These reports appear to involve traits which are either (1) unstable or inconsistently expressed, (2) possible artifacts of the sampling method (mushrooms too immature to provide representative data), or (3) associated with aberrant gross sporocarp morphologies which are unsuitable for the commercial market. In any event, although a long-felt need exists for increasing the production of homokaryons in breeding stock of Agaricus bisporus, none of the foregoing reports have led to a useful method for addressing this need.
The characteristic of producing only two spores on each basidium is disadvantageous to the mushroom breeder. Following meiosis in a typical two-spored basidium of Agaricus bisporus, each spore receives two nuclei which are jointly necessary for fertility. As a result, most spores of this species of mushroom, as historically known, produce fertile, heterokaryotic progeny. Such a trait of self-fertility poses a problem for the mushroom breeder because heterokaryons apparently undergo little, if any, hybridization. The bisporic trait characterizes all commercially cultivated strains as well as the great majority of naturally occurring or "wild" strains of Agaricus bisporus thus far discovered.
In contrast, all other known species of Agaricus produce predominantly fourspored basidia. In most agaric fungi, tetrasporic basidia are usually associated with the production of mononucleate homokaryotic spores which germinate to produce infertile homokaryons. In mushroom breeding, homokaryons, haploid strains which function in a manner similar to the gameres of plants and animals, are generally required for the practical crossbreeding of stocks to produce new hybrid strains. Homokaryons mate easily with other compatible homokaryons. However, less conventionally, it is sometimes possible to cross a homokaryon and a heterokaryon or, in some instances, to cross two heterokaryons.
To produce hybrids in the conventional manner, homokaryons such as those obtained from the homokaryotic spores of the parent varieties of mushrooms must fuse and establish a common heterokaryotic cytoplasm. However, homokaryons are presently very difficult to obtain by conventional spore isolation from the two-spored Agaricus bisporus strains because typically less than three percent of the spores produced by such strains are homokaryotic. The great majority of spores of these strains produce fertile, heterokaryotic progeny as noted hereinabove.
Moreover, heterokaryotic and homokaryotic offspring are generally indistinguishable from one another except by genetic screening, such as by the use of allozyme or DNA markers, which is time consuming and costly. Homokaryons are also difficult to obtain by other presently available methods. For a more complete description of some conventional methodologies for the recovery of Agaricus bisporus homokaryons, and some difficulties and drawbacks thereof, see "Strategies For The Efficient Recovery of Agaricus bisporus Homokaryons" by Kerrigan et al. in Mycologia, 84(4), 575-579 (1992), hereby incorporated by reference.
In order to overcome the difficulties associated with obtaining homokaryons from the two-spored Agaricus bisporus strains, attempts have been made to interbreed a four-spored strain from another known species of Agaricus with a two-spored strain of Agaricus bisporus. For example, in Raper, "Sexuality and Life Cycle of the Edible, Wild Agaricus bitorquis," Journal of General Microbiology, 95, 54-66 (1976) a homokaryon from the species Agaricus bitorquis was crossed with a homokaryon from the species Agaricus bisporus. However, Raper was unable to establish stable, fertile heterokaryons, and thus, was precluded from cross-breeding. In fact, no attempt to interbreed a four-spored species of Agaricus with a two-spored Agaricus bisporus has ever been successful. Accordingly, heretofore, any very highly four-spored strain of Agaricus was believed not to belong to the species Agaricus bisporus and was believed not capable of interbreeding with the species Agaricus bisporus.
Nevertheless, the need has remained for a process which will permit the mushroom breeder to obtain relatively large percentages or fractions of homokaryons relatively quickly, efficiently and inexpensively from the breeding stock of the Agaricus bisporus mushroom. Moreover, the mushroom breeder has always strived to increase the mushroom productivity yield and to shorten the duration of the mushroom crop cycle. A crop of mushrooms which are higher yielding and earlier fruiting than most commercial mushrooms would be economically desirable.